Covalently-bound conjugates, comprising an active drug and a carrier, are useful as a means of delivering the drug, e.g. to a specific site of action. Albumin has been proposed as a carrier for this purpose. Microparticles of albumin, their production and use as a carrier, are described in WO-A-9618388.
The covalent attachment of large peptides and proteins to human serum albumin (HSA) microcapsules can generate a number of problems. Sufficient binding sites may not be available for cross-linking due to poor contact between the protein and the microcapsule surface. Also, intra-rather than intermolecular cross-linking is difficult to control when using short or zero length cross-linkers such as glycolaldehyde or EDC, respectively. This can lead to low loading of microcapsules and inactivation of the protein.
Agam and Livne, in a series of papers, Blood 55:186-191 (1983), Thromb. Haemostasis 51:145-9 (1984) and 59:504-6 (1988), and Eur. J. Clin. Invest. (1991), showed that fibrinogen coated on fixed platelets augmented platelet aggregation, and that fibrinogen-coated erythrocytes reduced bleeding times in thrombocytopenic rats. Fixation involved the use of formaldehyde.
Coller et al, J. Clin. Invest. 89:546-555 (1992), describe "Thromboerythrocytes", an autologous, semi-artificial alternative to platelet transfusions. In order to avoid the limitations and drawbacks of using fresh platelets, erythrocytes were coupled to peptides containing the RGD cell recognition sequence, using a bifunctional cross-linker.
In an abstract presented to the XVth Congress of the International Society on Thrombosis and Haemostasis, Jerusalem, Israel, on Jun. 11-16, 1995, Yen et al reported the haemostatic potential of "Thrombospheres", i.e. cross-linked HSA microspheres, mean diam. 1.1-1.3 .mu.m, with human fibrinogen covalently bound to their surfaces. In a thrombocytopenic rabbit model, ear bleeding times were reduced. The HSA microspheres were reportedly prepared by the procedure described in U.S. Pat. No. 5,069,936, i.e. a solution/desolvation process using glutaraldehyde as a cross-linking agent, ethanol to cause precipitation, and a surfactant to modify the surface of the cross-linked protein molecules. These steps do not provide size control of the microspheres, may cause bound protein to be degraded, and are unsuitable for large-scale manufacture of platelet substitutes.
U.S. Pat. No. 5,069,936 describes the covalent bonding of various biological molecules, but not fibrinogen. A polyaldehyde is proposed as a covalent linking agent. Examples 12 and 14 use glutaraldehyde to bind antibody and enzyme (alkaline phosphatase), respectively.
WO-A-9639128 (published Dec. 12, 1996) also describes "Thrombospheres". Again, no specific preparation is given.
Fibrinogen is an adhesive glycoprotein containing the sequence RGD(S). It and other such glycoproteins (including fibronectin and collagen, among others) may mediate the adhesion of tumour cells to sub-endothelial layers. These glycoproteins interact with integrins found in tumour cells, e.g. the fibronectin receptor, and the GPIIb/IIIa receptor on platelets; see Dardik et al, Int. J. Cancer 70:201-7 (1997).
One of the major problems in the surgical treatment of cancers is the increased risk of tumour cells being released into the circulatory system. This is one reason for increased morbidity in patients with prostate cancer, after surgery. It would be desirable to remove circulating metastatic tumour cells, or inhibit their deposition on vascular surfaces.